The present invention addresses the problem of determining information concerning the conformation of biomolecules and changes in the conformation of biomolecules which result from an interaction with a chemical species, such as a biological macromolecule or ligand.
The conformation of biomolecules affects, and is affected by, their chemical interactions. For example, protein binding affects the shape and activity of DNA; protein-DNA bending has been shown to facilitate the assembly of nuclear proteins complexes and play a fundamental role in the control of transcription and replication. Conversely, the intrinsic bending of DNA and the inherent deformability associated with specific base sequences can affect protein recognition and binding. The extent to which DNA is bent intrinsically and on interaction with proteins is the subject of ongoing study. Rapid methods for the detection of DNA bending would facilitate this process. Rapid methods for the detection of DNA bending would facilitate screening for potential drugs that have a mode of action involving a change in DNA conformation, for example, drugs which affect transcription and regulation by affecting protein-driven bending.
Although the invention will be discussed further with reference to the determination of information concerning the conformation of double stranded DNA, and changes in the conformation of double stranded DNA resulting from interactions of the double stranded DNA, the invention is relevant to the determination of information concerning the conformation of other biological macromolecules, such as proteins, RNA, ssDNA etc. and changes in their conformation resulting from interactions.
Commonly employed methods for measuring DNA curvature include electrophoretic mobility assays and cyclisation assays based on measurements of the rate at which DNA can form enzymatically sealed closed circles. However, it can be difficult to interpret the results of these assays. Three-dimensional crystal structures of DNA-protein complexes provide detailed insight into the mechanism of protein-driven DNA bending, but this information is only available after a long and labour-intensive process. Solution-based structural analysis via NMR will likewise provide detailed information, but such methods are not applicable to all cases and are not suitable for rapid assays. Structural information can be obtained from atomic force microscope images, but the resolution is poor.
End-tethered DNA has been studied by fluorescence to characterise the conformation of both small and large DNA molecules. Confocal microscopy of large DNA molecules with intercalated dye has been used to provide evidence that the radius of gyration of end-tethered molecules is the same as that for molecules in solution. Fluorescence interference measurements with short DNA end-labelled with a fluorophore give a measure of the height of the fluorescent label within the DNA later, which can provide indirect evidence as to the tilt of end-labelled DNA, the shape of single-stranded DNA and the extent of hybridisation. However, a disadvantage of these techniques is that they require a label and provide only limited information concerning conformation.
Accordingly, the invention aims to provide a method of determining information concerning the conformation of biomolecules and changes in the conformation of biomolecules which result from an interaction with an agent (such as another biomolecule or a chemical entity), which is suitable for label-free sensing. The invention can be used for rapid and/or parallel screening, although it may also be used to study the conformation of a specific molecule or the change of conformation of a specific molecule following a specific interaction. Some embodiments of the invention provide real-time information concerning conformation.
Although the invention will be discussed further with reference to the determination of information concerning the conformation of biomolecules and changes in the conformation of biomolecules resulting from interactions of the biomolecules using a shear acoustic wave sensor, the invention may be performed using other types of liquid-phase acoustic wave sensor. By a “liquid-phase acoustic wave sensor” we mean an acoustic wave sensor in which the sensing surface of the acoustic wave sensor is in contact with a liquid in use.
It is known to use a shear acoustic wave sensor to investigate the properties of layers of material which are adhered to a sensing surface of the sensor. Shear acoustic wave sensors probe the response of a thin layer attached to the device surface to a mechanical displacement and are, thus, sensitive to the mechanical properties of the layer and the liquid medium which is within the penetration depth of the acoustic wave of the sensing surface. The interaction between shear mode acoustic waves and continuous, firmly attached elastic layers, such as metal films and/or homogenous viscous solutions has been comprehensively described both theoretically and experimentally. The viscosity of a surface layer of biomolecules will be dependent on, amongst other things, the conformation of the biomolecules, and it is known to determine the viscosity of a surface layer which is attached to the sensing surface of a shear acoustic wave device.
However, to date, biomolecules attached to the sensing surface of a shear acoustic wave device have been analysed as if they formed a homogenous viscoelastic layer where the measured signal includes contributions from both immobilised biomolecules and solvent molecules which are trapped in-between immobilised biomolecules. Viscoelastic layers have been modelled using the simple Maxwell or Voigt mechanical models for a viscoelastic layer, in which the layer is treated as being composed of elastic springs and viscous dashpots. These models have been used to derive information related to the viscosity and shear modulus of the film without making any quantitative reference to the specific conformation of the biomolecules within the sensed film.
The invention aims to provide a novel approach to using liquid-phase acoustic wave sensors to investigate the conformation of biomolecules, and changes in the conformation of biomolecules as a result of interactions, and a novel approach to analysing the signals produced by liquid-phase acoustic wave sensors, which facilitates the investigation of the conformation of biomolecules, and changes in the conformation of biomolecules as a result of interactions.
Within this specification and the appended claims, references to proteins, RNA, DNA or other biological macromolecules are intended to include both natural macromolecules and synthetic variants, such as proteins including non-proteinogenic residues, polynucleic acids including non-natural bases etc. The term “protein” is not intended to imply any specific minimum number of peptide residues.